Pharmaceutical composition of F(ab&#39;)2 antibody fragments

ABSTRACT

The present invention is directed to a pharmaceutical composition comprising F(ab′) 2  antibody fragments that are preferably free from albumin and of whole antibodies and also substantially free of pyrogens, and an effective amount of a pharmaceutically acceptable carrier. It is also directed to a method for the production of a pharmaceutical composition comprising F(ab′) 2  antibody fragments using serum or blood plasma of a mammal that has been previously immunized as a source of antibodies. The serum or blood plasma is digested with an enzyme pepsin, followed by separation and purification until the pharmaceutical composition of F(ab′) 2  fragments is free of albumin and complete antibodies, and substantially free of pyrogens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional patent application and claims priorityunder 35 U.S.C. § 120 of U.S. patent application Ser. No. 09/798,076,filed Mar. 5, 2001, now U.S. Pat. No. 6,709,655.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a pharmaceutical compositioncomprising F(ab′)₂ antibody fragments that are preferably free fromalbumin and of whole antibodies and also substantially free of pyrogens,and an effective amount of a pharmaceutically acceptable carrier. It isalso directed to a method for the preparation of a pharmaceuticalcomposition comprising F(ab′)₂ antibody fragments using serum or bloodplasma of a mammal that has been previously immunized as a source ofantibodies. The serum or blood plasma is digested with an enzyme,pepsin, followed by separation and purification until the pharmaceuticalcomposition of F(ab′)₂ fragments are free of albumin and completeantibodies, and substantially free of pyrogens.

2. Background Art

Antibodies are proteins of a globulin type known as immunoglobulins thatare present in blood serum as a response of the immune system to theinvasion of some foreign substance or organism, and are characterizedfor specifically combining with those substances that are foreign to theorganism, neutralizing them and precipitating them so that they areremoved from circulation. Various industrial applications have beendeveloped with them for the diagnosis, monitoring, prevention andtreatment of different ailments.

In regions where, due to climatic conditions, venomous animals abound,antibodies have been given a special use to combat venom. A large numberof doses are applied when treating patients with scorpion, spider andsnake stings or bites, principally. At present, a use that is gaining inimportance is as a treatment for auto-immune diseases like rheumatoidarthritis, immune-dependent diabetes mellitus, AIDS, hemophilicanaemias, rheumatic fever, multiple sclerosis, thyroiditis andpsoriasis, among others. In these cases, anti-cytokine antibodies areapplied either directly to the patient or by treating blood that hasbeen taken from and is subsequently re-fed to the patient, in order toremove the cytokines generated by the organism itself in response to theailment. If such cytokines are not removed, they will cause extremelytroublesome symptoms (see U.S. Pat. Nos. 5,888,511 and 4,940,670).

There are several kinds of immunoglobulins, known as IgG, 1 gM, IgD, IgAand IgE, of which IgGs are the most abundant in the blood circulation.IgGs correspond to a mature immune response and therefore include thevast majority of antibodies that are commercially produced. All the IgGshave the same general structure (which can be seen in FIG. 1). They arecomposed of four polypeptide chains, two that are heavy (H) and twolight (L), which are joined together by disulfide bridges. The two heavychains, in turn, are joined together by two other disulfide bridgesknown as the hinge region, approximately halfway along the chains. Alittle closer to the amino terminal region, each heavy chain is joinedby a disulfide bridge with a light chain. Each heavy chain has threeconstant regions, C_(H)1, and C_(H)2, and C_(H)3, the last two in thecarboxy terminal region (before the hinge) and the first in the aminoterminal region (immediately after the hinge) and a Variable region (VH)in the amino terminal end, while each light chain has only one constantregion, CL, in the carboxy terminal end and one variable region, VL, inthe amino terminal end.

When the IgG is digested enzymatically, different fragments are obtaineddepending on the enzyme used, that is, if papain is used, threefragments are obtained, the crystallizing fragment (Fc) and twoantigen-binding fragments (Fab) and, if pepsin is used, one F(ab′)₂fragment is obtained, while the crystallizing fragment is digested. Theforegoing is due to the fact that papain cuts the heavy chainsimmediately after the hinge (towards the amino terminal region), whilepepsin cuts them before the hinge (towards the carboxy terminal region).Fab and F(ab′)₂ fragments conserve their capacity to specifically bindto the antigen that gave rise to them. F(ab′)₂ fragments alsoprecipitate antigens, while the Fc antibody fraction normally acts as amarker signal for macrophages as well as the activation of lymphocytesfor the recognition and phagocytosis of the antigen-antibody complex.

The Fc fragment comprises the antigenic determinants of the antibody insuch a way that when a patient is administered whole antibodiesgenerated in some animal of another species, the patient generates animmune response against these antigenic determinants. This may give riseto varied adverse secondary responses that can even include anaphylacticshock.

These problems are significantly reduced when the antibodies arepreviously digested with papain or pepsin and only the resultingpurified Fab or F(ab′)₂ fragments are administered.

The use of Fab or F(ab′)₂ fragments has another advantage that is knownas the concept of distribution volume, which is simply the volume of thebody in which a determined drug is dissolved. This volume can refer tothe circulating blood alone, as is the case of IgG, or can include alarger part of body water in the case of the fragments. For this reason,as Fab and F(ab′)₂ have a greater corporeal volume they can neutralizetoxins lodged in various tissues, not only in the blood. They can evencross the blood/brain barrier in both directions and be used toneutralize or eliminate neurotoxins.

The use of F(ab′)₂ fragments has a particular advantage over the use ofFab fragments in that they are retained far longer in the organismbecause they have double the molecular weight. Moreover, they conservetheir capacity to precipitate the antigen in physiological conditions aswell as maintaining a size that allows them access to a distributionvolume that is sufficient for treatment purposes.

Due to the fact that the F(ab′)₂ fragments conserve the maincharacteristics of the antibodies, the applications of the antibodiesextend to F(ab′)₂ fragments, with the additional advantage that becausethey lack the Fc fragment, recognition as foreign by a patient to whomthey are administered is less likely. This provides greater tolerance toapplication of F(ab′)₂ fragments and reduces the possibility ofsecondary reactions, which is particularly useful for prolongedtreatments such as those applied in autoimmune diseases.

It has been known for many years that soluble proteins (particularlyserous proteins) lose solubility as the concentration of neutral salts(such as ammonium and sodium sulfates) in the solution increases. Inthis way, for example, euglobulin precipitates with 13.5% sodiumsulfate, pseudoglobulin with 17.4% and pseudoglobulin 2 with 21.3%. Thisfact has been used to partially purify antibodies from serum or plasma.

Several approaches in the production of antibodies and their fragmentshave been reported in the literature. For example, U.S. Pat. No.4,849,352, to Sullivan et al., claims the production of both Fabfragments through the digestion of antibodies with papain immobilized inpolyacrylamide. Sullivan et al. also claims the production of F(ab′)₂fragments through the digestion of antibodies with immobilized pepsin,obtaining Fab and Fc or F(ab′)₂ fragments and subsequently purifying thefragments through immunoaffinity, passing them through a polyacrylamidesieve containing the specific antigen of the antibodies in question.Later the Fab or F(ab′)₂ fragments that have specifically bound to themolecules in the sieve are recovered with some strongly ionic solution.The use of immobilized enzymes for digestion and immobilized antigensfor purification could prove to be extremely expensive for the largescale commercial production of preparations of antibody fragments, whichis a drawback despite the purity of the fragments obtained.

Furthermore, although an antigenic sieve may be useful for producingantibody fragments against pure substances, this method is noteconomically feasible to produce antibodies against venoms that aremixtures of a large number of toxins, many of which have a biologicaleffect.

Another approach is shown in U.S. Pat. No. 5,733,742 in which Landonclaims a process to produce Fab fragments using whole blood in a sterilemedium, in which the whole blood is put directly into contact with theenzyme, free or immobilized, that has preferably been purified.Subsequently, the cell residues are removed by centrifugation,separating and recovering the resulting fragments that are subsequentlypurified preferably by immunoaffinity. Again, Landon used purifiedantigens which, unlike venoms, can easily be bound to supports to obtaina sieve for the purification of the Fab of interest. Landon never usedor discussed the method of obtaining Fab fragments against antigens thatare mixtures of many substances, as is the case of venoms. He onlyworked with papain and chemopapain and did not discuss the possibilityof using pepsin.

An additional approach to the production of Fab fragments is shown inU.S. Pat. No. 4,814,433 in which Fredrickson describes a procedure forobtaining papain free Fab. He observes that when antibodies are digestedby this enzyme some contaminants remain in the solution. Thesecontaminants are hybrid compounds of the papain joined by disulfidebridges to some of the fragments resulting from the digestion, which thepapain can subsequently continue digesting and degrading the fragmentsobtained. In order to solve the problem, Fredrickson used antipapainantibodies, which capture the hybrid compound of the enzyme.Subsequently, the fragments were purified by passing the solution alonga column with protein A in which the Fc fragments and the hybridcompounds were retained. This problem, present in the digestion withpapain, has not been reported when digestion is done with pepsin.

Some traditional methods involve the digestion of pepsin and theprecipitation of the fraction of the fragments with ammonium or sodiumsulfates, but a pre-separation is usually done with the antibodies byprecipitation with sulfate and then digestion of the antibody fraction.However, large losses have been reported of the biological activity inthe resulting fragments and a high content of intact antibodies andother contaminants.

As can be seen from the background, although there are several methodsfor the production of Fab antibody fragments, they are often difficultto apply as immunogens in the case of complete venoms. Furthermore, theadvantage of using F(ab′)₂ fragments in this case is clear since theyhave a greater retention time than Fab and do precipitate neutralizingtoxins. Moreover, the reports of F(ab′)₂ fragment production by means ofdigestion with pepsin have given evidence of a considerable loss ofbiological activity and a high content of whole antibodies and otherimpurities, which has discouraged the commercial production ofpharmaceutical products comprising this type of fragment.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention relates to a pharmaceutical compositioncomprising F(ab′)₂ antibody fragments that are preferably free fromalbumin and of whole antibodies and also substantially free of pyrogens.

Another aspect of the present invention is directed to a pharmaceuticalcomposition comprising F(ab′)₂ antibody fragments in an effective amountof a pharmaceutically acceptable carrier.

In still another aspect of the invention, the invention provides apharmaceutical composition comprising F(ab′)₂ antibody fragments whichneutralizes or eliminates toxins in tissues and blood.

Another aspect of the invention relates to a pharmaceutical compositioncomprising F(ab′)₂ antibody fragments which neutralizes a complexmixture of antigenic molecules such as the venom of venomous animal.

Another aspect of the present invention is directed to a method for thepreparation of a pharmaceutical composition comprising F(ab′)₂ antibodyfragments using the serum or blood plasma of a mammal that has beenpreviously immunized as a source of antibodies. The serum or bloodplasma is digested with an enzyme, pepsin, followed by separation andpurification until the pharmaceutical composition of F(ab′)₂ fragmentsare free of albumin and complete antibodies, and substantially free ofpyrogens.

Other aspects of the present invention will be apparent to one ofordinary skill on consideration of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 is a general structure of an Antibody. H represents a heavychain. L represents a light chain, C represents a constant regionwhether in a heavy or in a light chain and V represents a variableregion whether in a heavy or in a light chain. HiR represents the hingeregion.

FIG. 2 is a diagram of F(ab′)₂ production. The main stages of theprocess of the present invention for the production of F(ab′)₂ fragmentsare outlined in the Figure. Blocks I to XII represent: Hyperimmune bloodplasma mixture (I); Digestion of the plasma (II); Precipitation ofundesired protein fragments (III); Clarification of the F(ab′)₂ solution(IV); Waste containing undesired protein fragments precipitate (V);partially purified F(ab′)₂ solution (VI); precipitation of F(ab′)₂ fromsolution (VI); Centrifugation (VIII); Waste containing low molecularcomponents and salts (IX); Dialysis or ultrafiltration (X); sterilefiltration and formulation (XI); Lyophilization (XII); Final product(XIII).

FIG. 3 is a general structure of an F(ab′)₂ fragment. H represents aheavy chain fragment. L represents a light chain. C represents aconstant region whether in a heavy chain fragment or in a light chainand V represents a variable region whether in a heavy chain fragment orin a light chain. HiR represents the hinge region.

FIG. 4 is an electrophoresis of the different stages of the process forobtaining F(ab′)₂ against polyvalent scorpion venom. Lanes 1 and 9correspond to molecular weight markers: Myosin (205 Kd), R-galactosidase(121 Kd), Bovine serum albumin (70 Kd), Ovolbumin (52.4 Kd), Carbonicanhydrase (34.9 Kd), Soybean Trypsin Inhibitor (29.1 Kd), Lysozyme (20.7Kd) and Aprotinin (6.9 Kd); Lanes 3 to 8 represent: Blood plasma,digested plasma, the mixture from the first precipitation, filtrate,waste and mixture from the second precipitation and Lanes 11 to 16represent: precipitate paste, dialysis, waste, raw F(ab′)₂(Concentrated), Sterile formulated F(ab′)₂ solution and Final product.Gels 1 and 2 are under non-reducing conditions and gels 3 and 4 areunder reducing conditions.

FIG. 5 is an electrophoresis of different lots of F(ab′)₂ fragmentsagainst the venom of the black widow spider. Lane 1 corresponds tomolecular weight markers: Myosin (205 Kd), β-galactosidase (121 Kd),Bovine serum albumin (70 Kd), Ovalbumin (52.4 Kd), Carbonic anhydrase(34.9 Kd), Soybean Trypsin Inhibitor (29.1 Kd), Lysozyme (20.7 Kd) andAprotinin (6.9 Kd). Lanes 2-7 are different lots of F(ab′)₂ fragmentsagainst the venom of the black widow spider.

FIG. 6 is an electrophoresis of different lots of F(ab′)₂ fragmentsagainst the venom of the coral snake. Lane 1 corresponds to molecularweight markers: Myosin (205 Kd), β-galactosidase (121 Kd), Bovine serumalbumin (70 Kd), Ovalbumin (52.4 Kd), Carbonic anhydrase (34.9 Kd),Soybean Trypsin Inhibitor (29.1 Kd), Lysozyme (20.7 Kd) and Aprotinin(6.9 Kd). Lanes 2-5 are different lots of F(ab′)₂ fragments against thevenom of the coral snake.

FIG. 7 is an electrophoresis of different lots of F(ab′)₂ fragmentsagainst the venom of snakes of the Bothrops, Crotalus and Lachesisgenera. Lanes 1-9 are different lots of F(ab′)₂ fragments against thevenom of snakes of the Bothrops, Crotalus and Lachesis genera; whilelane 10 corresponds to molecular weight markers: Myosin (205 Kd),β-galactosidase (121 Kd), Bovine serum albumin (70 Kd), Ovalbumin (52.4Kd), Carbonic anhydrase (34.9 Kd), Soybean Trypsin Inhibitor (29.1 Kd),Lysozyme (20.7 Kd) and Aprotinin (6.9 Kd).

FIG. 8 is an electrophoresis of different stages of the process forobtaining F(ab′)₂ against cytokines. Lane 1 corresponds to molecularweight markers: Myosin (205 Kd), β-galactosidase (121 Kd), Bovine serumalbumin (70 Kd), Ovalbumin (52.4 Kd), Carbonic anhydrase (34.9 Kd),Trypsin Inhibitor (29.1 Kd), Lysozyme (20.7 Kd) and Aprotinin (6.9 Kd).Lane 2 is empty and Lanes 3 to 8 contain blood plasma, digested plasma,filtrate, filtrate waste, paste obtained from centrifugation andconcentrate after dialysis. Gels 1 and 2 correspond to F(ab′)₂anti-TNF-α under non-reducing and reducing conditions. Gels 3 and 4correspond to F(ab′)₂ anti interferon-γ under non-reducing and reducingconditions.

DETAILED DESCRIPTION OF THE INVENTION

The following glossary is provided as an aid to understand certain termsherein. The explanation provided in the glossary are for illustrativepurposes and do not limit the scope of the invention.

The term “substantially free” refers to the absence of pyrogen andprotein material foreign to F(ab′)₂ such as albumin or whole antibodiesin accordance with the standards of the Mexican Pharmacopeia.

The term “aseptic conditions” refer to precautionary measures or methodsemployed in handling the different products from each step of the methodof the present invention to prevent the contamination of culture orsterile media and infection by extraneous microorganisms.

The term “effective amount” or “pharmaceutically effective amount” of acompound in unit dose of the composition depends upon the number offactors. Included among these factors are quantity of the otheringredients when used and tolerance of the active ingredient ofcomposition. Effective amount of the active ingredient ranges from about8% to about 35% by weight based on the total weight of the composition.The amount of F(ab′)₂ preparation to be filled in each flask variesdepending upon the specie from which the venom was prepared. Forcompositions against scorpions, the F(ab′)₂ preparation to be filled ineach flask is the amount necessary to neutralize from about 135 to about220 lethal doses 50% of the venom. For compositions against black widowspider, the amount necessary to neutralize is from about 540 to about880 lethal doses 50% of the venom. For compositions against coral snake,the amount necessary to neutralize is from about 360 to about 660 lethaldoses 50% of the venom. For compositions against Bothrops and Crotalus,the flasks are filled with the amount necessary to neutralize from about700 to about 1100 lethal doses 50% of the venom.

By “pharmaceutically acceptable carrier” is meant solid or liquidfiller, diluent or substance which may be safely used in systemic ortopical administration. Depending on the particular route ofadministration, a variety of pharmaceutically acceptable carriers wellknown in the art include solid or liquid fillers, diluents, hydrotropes,surface active agents, and encapsulating substances. The amount ofcarrier employed in conjunction with the F(ab′)₂ fragments is enough toprovide a practical quantity of material per unit dose of composition.

Pharmaceutically acceptable carriers for systemic administration thatmay be incorporated in the composition of the invention include sugar,starches, cellulose, vegetable oils, buffers, polyols and alginic acid.Specific pharmaceutically acceptable carriers are described in thefollowing documents, all incorporated herein by reference: U.S. Pat. No.4,401,663 to Buckwalter et al., issued Aug. 30, 1983; European PatentApplication No. 089710, LaHann et al., published Sep. 28, 1983; andEuropean Patent Application No. 0068592, Buckwalter et al., publishedJan. 5, 1983. Preferred carriers for parenteral administration includepropylene glycol, pyrrolidone, ethyl oleate, aqueous ethanol, andcombinations thereof.

Representative carriers include acacia, agar, alginates,hydroxyalkylcellulose, hydroxypropyl methylcellulose,carboxymethylcellulose, carboxymethylcellulose sodium, carrageenan,powdered cellulose, guar gum, cholesterol, gelatin, gum agar, gumarabic, gum karaya, gum ghatti, locust bean gum, octoxynol 9, oleylalcohol, pectin, poly(acrylic acid) and its homologs, polyethyleneglycol, polyvinyl alcohol, polyacrylamide, sodium lauryl sulfate, poly(ethylene oxide), polyvinylpyrrolidone, glycol monostearate, propyleneglycol monostearate, xanthan gum, tragacanth, sorbitan esters, stearylalcohol, starch and its modifications. Suitable ranges vary from about0.5% to about 1%.

In accordance with the above, the present invention is related to apharmaceutical composition that comprises F(ab′)₂ antibody fragmentsthat are free of albumin and whole antibodies and substantially free ofpyrogens. The antibodies from which the fragments are obtained can begenerated against purified molecules, i.e., free of other antigenicmolecules. Examples of purified molecules include cytokines,particularly TNF-α and interferon-γ or other pharmaceutical drugs andthe like. The antibodies from which the fragments are obtained can alsobe generated against complex mixtures of immunogenic molecules such asthe venoms of poisonous animals that are complex mixtures of peptidesand toxins, and even a mixture of the venoms of several species. Moreparticularly, the pharmaceutical composition of the present inventionhas been successfully obtained using antibodies generated against thecytokines TNF-α and interferon-γ, against the total venom of the blackwidow spider (Latrodectus mactans), the total venom of the coral snake(Micrurus nigroscinctus), against the venoms of snakes of the generaBothrops, Crotalus, Agkistrodon, Lachesis and Sistrurus and against amixture of venoms (polyvalent venom) of scorpions, particularlyCentruroides noxius, C. limpidus limpidus, C. limpidus tecomanus and C.Suffussus sufjussus. The pharmaceutical composition of the presentinvention comprises F(ab′)₂ fragments, the general structure of which ispresented in FIG. 3, that lack the Fc region, are free of albumin andcomplete antibodies, and lack antigenic determinants that would make thepatient's organism recognize them as foreign. In this way, thepharmaceutical composition of this invention can be administered on morethan one occasion, dramatically reducing the possibility of a secondaryresponse. The administration is normally systemic administration,whether intramuscular or intravenous. The amount varies on thecharacteristics of the subject to be administered, the specie that bitor stung the subject, and the degree of administration. For example, forscorpions or black widow spiders, about 1 to about 3 flasks are employedwhile about 1 to about 10 flasks are used for snakes.

Similarly, this invention is aimed at a method for the production ofF(ab′)₂ antibody fragments, substantially free of albumin, wholeantibodies and pyrogens from a source of antibodies such as serum,plasma or the blood of some animal which has been subjected to animmunization scheme with an immunogen, stimulating the generation ofspecific antibodies against the immunogen.

Obtaining a source of antibodies of a good quality, that is with a hightiter of antibodies with high specificity against the immunogen and ahigh degree of representation of the possible different antibodies thatcan recognize the various antigenic determinants of the immunogen, isbeneficial to obtain F(ab′)₂ antibody fragments substantially free ofpyrogens and protein material foreign to said fragments.

The initial material, that is the antibody source, should preferablycome from several animals in order to have a greater universe ofdifferent specific antibodies against the different epitopes of theimmunogen. The antibody source can be blood, serum or preferably plasma.

With the purpose of obtaining a product substantially free of pyrogens,extremely rigorous conditions of asepsis must be maintained during thewhole process, although it is not necessary to have a closed sterilesystem during the whole process as established by Landon in U.S. Pat.No. 5,733,742.

The first step in the method is the digestion of the protein materialcontained in the antibody source, although dilution of the antibodysource is recommended as an option, especially in the case of plasma. Todo so, the pH is lowered to about 3.2±0.2 and sufficient amounts of theenzyme pepsin (from about 0.5 to 1 g/l depending on the enzyme activity)are added. This mixture is incubated with agitation for the intervals oftime needed to obtain a high degree of hydrolysis (close to 100% of thehydrolyzed antibodies) at a temperature of nearly 20° C.

Precipitation is then conducted by adding ammonium sulfate in aproportion of between about 16 and about 22% (W/V), preferably about21%, incubating the mixture for at least 30 minutes at a temperature ofabout 55± about 4° C. The mixture is subsequently cooled and left tosettle at a temperature of between about 8 and about 12° C. for at least2 hours. This settling period is of great importance in the formation ofparticles of a larger size, which results in greater precipitationefficiency, and the absorption of other impurities of a lesser size thatwould not precipitate otherwise. In this step, most of the undigestedserum proteins such as albumin and fibrinogen and the large fragmentsresulting from theft digestion are precipitated, while the F(ab′)₂fragments resulting from the digestion of the antibodies remain in thesolution.

The following step comprises clarifying the solution by eliminating thesmallest particles formed during the precipitation. To do so, the use of12, 8 and 4, and optionally 0.22 μ, tray filters is suggested to reducethe possible presence of pyrogens. F(ab′)₂ fragments and some solublepeptides produced by the degradation of the albumin and fibrinogen canbe found in the recovered supernatant.

The supernatant from the foregoing step is submitted to a furtherprecipitation by adding anmmonium sulfate in a proportion between about32 and 38%, preferably about 35% Weight/Volume at a pH of about 6.8±0.5.Again, it is important to allow for the longest settling periodpossible, in refrigeration, ideally for at least 12 hours, with whichlarger particles with greater precipitation efficiency are obtained. Thesalts and some components of a low molecular weight that do notprecipitate in the conditions handled in the previous steps remain insoluble form in the supernatant.

In order to separate the particles from the supernatant, it isrecommended to centrifuge the suspension formed at between about 10,000and 15,000 rpm, recovering a paste of precipitated F(ab′)₂ fragments.

With the purpose of purifying the produced, isolated F(ab′)₂ fragments,it is necessary to remove the salts and components of a low molecularweight that have been captured in the precipitation. To do so, thecrystal paste can be submitted to dialysis or alternatively toultrafiltration. In both cases, the membrane pore must have a size thatpermits the salts and components of a low molecular weight to passthrough it, but not the F(ab′)₂ fragments, which become soluble again asthe concentration of salts decreases.

In order to guarantee that the final product is substantially free frompyrogens, the solution formed in the previous step is passed through asterile filter and is subsequently formulated with pharmaceuticallyacceptable excipients for injectable substances. Polyvinylpyrrolidone,mannitol or dextrose may be used, Osmolytes (such as glycerol),stabilizers like saccharose, and some salts like NaCl may be used inorder to achieve isotony in the solution to be administered, dosifyingthe product so that each dose (flask) contains adequate potency.

The product is subsequently lyophilized and the flasks containing it arehermetically sealed. In this way, an easily soluble product is obtained(the content of one flask in 5 ml in less than 1 minute), which issubstantially free of pyrogens according to the Pharmacopoeia andprotein material foreign to F(ab′)₂ (0% albumin, 0% whole antibodies).

Methodology

Pyrogen Test

The test was conducted in accordance with the Pharmacopoeia of MexicoMGA 0711 Pyrogen Test.

Biological Potency Assay

The assay was conducted in accordance with the Pharmacopoeia of MexicoMPB 050. Anti-poison sera potency.

Electrophoresis SDS

Electrophoresis was conducted using 10% acrylamide gels, in bothreducing and non-reducing conditions. The protein concentration in eachsample was standardized at 18 μg per lane. They were developed withethidium bromide.

Solubility

Solubility was conducted by adding 5 ml of bidistilled water to eachflask and shaking vigorously and observing the solubility of the contentof the flask after less than one minute against the light.

Lethal Doses 50% Determination.

To determine the lethal doses 50% for every venom disclosed, eightserial dilutions (1:3) of every venom in saline solution were prepared.After that, eight groups of 5 mice (Balb/c) of an average weight of 15 gwere injected intraperitoneally with 100 μg of the dilutions, one groupfor each dilution of each venom. Mortality of mice was read after 24hours.

Experimental data of percentage of mortality vs. logarithm of venomdoses were adjusted by a non-lineal regression with GraphPad PRISMsoftware (GraphPad Software Inc., San Diego, Calif.) and the LethalDoses 50% (LD5O) were calculated for each venom as the dose at which 50%of the mice administered with such dose died.

ELISA Procedure for the Determination of Anti-Venom Titer.

1. The lyophilized venoms were reconstituted in 0.1M sodium carbonatebuffer pH 9.5 to a concentration of 5 μg/ml.

2. 96 wells ELISA plates were coated by adding 100 μl of venom per welland incubated overnight at 4° C.

3. Wells were washed three times with 200 μl/well of washing solution(50 mM Tris/HCI pH 8, 0.15 mM NaCl, 0.05% Tween 20).

4. Unspecific unions were blocked with blocking solution (50 mM Tris/HCIpH 8.0, 0.5% gelatin, 0.2% Tween 20) for 2 hours at room temperature.

5. Serial dilutions of antivenom with an initial stock of 1:10 were madein situ reaction buffer (50 mM Tris/HCI pH 8.0, 500 mM NaCl, 0.1 mg/mlgelatin, 0.05% Tween 20)

6. Thus, 100 μI of reaction solution (dilutions) were added to each welland 50 μl of the antivenom solution were mixed and then 50 μl of thedilution were transferred to the next well and so on until 10 wells, andwere incubated 1 hour at room temperature.

7. Again, wells were washed three times with 200 μl/well of washingsolution.

8. A second antibody against horse was added, which was conjugated tothe enzyme peroxidase diluted 1:2500 in reaction buffer and wasincubated at room temperature for 1 hour.

9. Reaction was developed by adding 100 μl well of the chromogenicsubstrate ABTS and incubating for 10 minutes at room temperature.

10. Reaction was stopped by adding 25 μl/well of concentratedfluorhydric acid.

11. Absorbance was read at 405 mn with an ELISA reader

EXAMPLES

In order to illustrate better the pharmaceutical compositions and themethod of the present invention for the production of F(ab′)₂ antibodyfragments, the following specific examples are provided to better assistthe reader in the various aspects of practicing the present invention.As these specific examples are merely illustrative, nothing in thefollowing descriptions should be construed as limiting the invention inany way.

Example 1 Development of an Adequate Source of Antibodies

In order to have an adequate source of antibodies, it is necessary toattempt to have the following requisites: a) high titer antibodies,implying a high concentration of antibodies and a high percentage ofsaid antibodies that are specific against the antigen in question and apopulation of wide spectrum antibodies, that is, against the differentepitopes of each of the molecules comprising the antigen; this isparticularly important in the case of venoms.

In order to obtain a high titer it is necessary to follow the followingrecommendations:

Adequately prepare and formulate (conjugations, use of aids) antigens tomaximize their immunogenic capacity.

Apply immunization schemes capable of efficiently activating theanimal's immune system.

Determine in each group of animals the moment when they reach each stageof the immune response curve (where they reach a peak and level off,mainly). This can be performed by means of neutralization assays.

Maintain a selection of high producer animals taking special care toprolong their average life span with respect to antibody production.

In order to obtain a population of wide spectrum antibodies, it isrecommended to use a mixture of blood, plasma or serum from differentanimals.

Immunization schemes like those recommended in the literature werefollowed with doses of venoms that ranged from 3 to 150 DL₅₀ per horsethroughout 12 immunizations given over 5 to 6 weeks for the baseschemes, and from 70 to 450 DL₅₀ per horse throughout 5 immunizationsover 3 weeks for the reinforcement schemes, according to the type ofvenom applied. Freund's Complete and Incomplete adjuvants were used aswell as a saline isotonic solution, using a total of 5, 10 or 20 ml inthe different inoculations.

The antibody source used in the present invention was obtained frombleeding animals, at a rate of 6 to 10 liters per horse perbloodletting, two bloodlettings during the 15 days following the lastinoculation of each scheme.

The antibody source for the method of the present invention can be amixture of blood from different animals immunized with the same antigen,or the serum produced by its coagulation, or preferably the plasmaresulting from the separation of the cell packet by sedimentation. Theparticular use of plasma has the advantage in large scale productionthat the cell packet can be washed and suspended in a physiologicalsolution and fed back to the animal from which it was obtained, thusreducing the stress associated with the drop in blood cells andproducing the smallest impact possible on antibody production.

In this way, antibody sources were produced against the venom of thescorpion (a polyvalent venom which is a mixture of the venoms of thescorpions Centruroides noxious, C. limpidus limpidus, C. limpidustecomanus and C. suffussus suffussus; of the black widow spider(Lactrodectus mactans); the coral snakes (Micrurus nigroscienctus); andsnakes of the genera Bothrop, Crotalus and Lachesis, particularly therattlesnake (Crotalus durissus durissus), the mute rattlesnake (Lachesismuta stenophry) and the nauyaca (Bothrops asper).

Example 2 Application of the Method of the Present Invention for theProduction of Polyvalent Anti-Venom Against Scorpion Venom

An antibody source was obtained as mentioned in Example 1, using in thiscase polyvalent scorpion venom (Centruroides noxius, C. limpiduslimpidus, C. limpidus tecomanus and C. suffussus suffussus) as antigen,obtaining blood plasma as antibody source. The plasma of differentanimals immunized against the same polyvalent venom was mixed together.

The plasma was diluted with depyrogenized water 1:2 (inverse osmosis,sterilized and filtered by 0.22μ and the pH adjusted at about 3.2.Pepsin (filtered by 0.22μ) was added for digestion until nearly 390,000units per liter were obtained and the mixture was left to react at atemperature close to 20° C. for about one hour, with agitation at about15 minute intervals.

Once the reaction was completed, the mixture was heated to about 54° C.and ammonium sulfate was added in a proportion of 21% (weight/volume)and it was left to settle for 30 minutes. The mixture was subsequentlyrefrigerated at a temperature of between about 4 and about 8° C. for aspace of about 2 hours (although it can be stored for up to about 24hours). The supernatant was recovered through decantation and clarifiedby passing it through tray filters of 12, 8 and 4μ.

Ammonium sulfate was again added to the clarified supernatant in aproportion of 35% (weight/volume), having previously adjusted the pH atabout 6.8. It was left to settle for 12 hours. The mixture wassubsequently centrifuged at some 15,000 rpm in a Sharples typecentrifuge. The recovered paste contains F(ab′)₂ fragments. The pastewas submitted to a process of dialysis in cellophane, at between about 4and about 12° C. for about 8 to 10 days. The resulting solutioncontained F(ab′)₂ fragments specific against scorpion venom that issubstantially pure and free of pyrogen.

Subsequently, the soluble part of that fraction that did not solubilizewas separated and formulated by adding saccharose, NaCl and glycerol,adjusting the pH at around 6.8 and it was dosified according to thedetermined potency in pyrogen free flasks that will then be lyophilized.

Tests for purity were conducted by Electrophoresis of different stagesof the process and HPLC with the results shown in FIG. 4. It can clearlybe seen that the F(ab′)₂ fragments produced have a molecular weight ofapproximately 100,000 to 110,000 daltons (non-reducing conditions),while under reducing conditions, they are present as bands of 25,000 to30,000 daltons. Similarly, potency tests were conducted showing that thepharmaceutical composition of the F(ab′)₂ fragments thus obtainedeffectively neutralize scorpion polyvalent venom. The content of all theflasks sampled were soluble in 5 ml of bidistilled water in less than 1minute.

Example 3 Application of the Method of the Present Invention for theProduction of Anti-Venom Against the Venom of the Black Widow Spider

A source of antibodies as described in Example I was then obtained,using the venom of the black widow spider (L. Mactans) and plasma waschosen as antibody source. The plasma obtained was processed in the sameway as in Example 2.

FIG. 5 shows the electrophoresis gel in different stages of the processwhere it can be clearly seen that the F(ab′)₂ fragments produced have amolecular weight of approximately 100,000 to 110,000 daltons(non-reducing conditions), while under reducing conditions, they arepresent as bands of 25,000 to 30,000 daltons. The potency test showedthat the pharmaceutical composition comprising the F(ab′)₂ fragmentsthus obtained effectively neutralizes the venom of the black widowspider. The contents of all the flasks sampled was soluble in 5 ml ofbidistilled water in less than 1 minute.

Example 4 Application of the Method of the Present Invention for theProduction of Antivenom Against the Venom of the Coral Snake

A source of antibodies as described in Example 1 was then obtained,using the venom of the coral snake (M. nigroscienctus) and plasma waschosen as antibody source. The plasma obtained was processed in the sameway as in Example 2.

FIG. 6 shows the electrophoresis gel in different stages of the processwhere it can be clearly seen that the F(ab′)₂ fragments produced have amolecular weight of approximately 100,000 to 110,000 daltons(non-reducing conditions), while under reducing conditions, they arepresent as bands of 25,000 to 30,000 daltons. The potency test showedthat the pharmaceutical composition comprising the F(ab′)₂ fragmentsthus obtained effectively neutralizes the venom of the coral snake. Thecontents of all the flasks sampled was soluble in 5 ml of bidistilledwater in less than 1 min.

Example 5 Application of the Method of the Present Invention for theProduction of Anti-Venom Against the Venom of Snakes of the Bothrops,Crotalus, Agkistrodon, Lachesis and Sistrurus Genera

A source of antibodies as described in Example 1 was then obtained,immunizing groups of animals separately with venom of snakes from eachof the following genera: Bothrops, Crotalus, Agkistrodon, Lachesis andSistrurus, one group for each venom, and plasma was chosen as antibodysource. The plasma obtained for each of the snake venoms of thedifferent genera was processed in the same way as in Example 2.

The F(ab′)₂ obtained for each of the snake venoms of the differentgenera is mixed, considering putting the same proportion in terms ofpotency for each of them, before being formulated and dosified inflasks. Subsequently, they are formulated with suitable excipients anddosified and lyophilized in flasks.

FIG. 7 shows the electrophoresis gel in different stages of the processwhere it can be clearly seen that the F(ab′)₂ fragments produced have amolecular weight of approximately 100,000 to 110,000 daltons(non-reducing conditions), while under reducing conditions, they arepresent as bands of 25,000 to 30,000 daltons. The potency test wasconducted in the individual F(ab′)₂ for each type of venom against therespective venom and subsequently in the mixture of F(ab′)₂ against eachtype of venom separately, showing that the pharmaceutical compositioncomprising the F(ab′)₂ fragments thus obtained effectively neutralizesthe venom of snakes of any of the genera Bothrops, Crotalus,Agkistrodon, Lachesis or Sistrurus. The contents of all the flaskssampled was soluble in 5 ml of bidistilled water in less than 1 minute.

Example 6 Application of the Method of the Present Invention for theProduction of Anti-Cytokines

Using blood plasma obtained from goats immunized with a cytokine (both(α-TNF and interferon-γ, separately) as source of antibodies, the sameprocedures were followed as in Example 2.

FIG. 8 shows the electrophoresis gels in different stages of the processwhere it can be clearly seen that the F(ab′)₂ fragments produced have amolecular weight of approximately 100,000 to 110,000 daltons(non-reducing conditions), while under reducing conditions, they arepresent as bands of 25,000 to 30,000 daltons. The potency test showedthat the pharmaceutical composition comprising the F(ab′)₂ fragmentsthus obtained effectively neutralizes the TNF-α and interferon-γcytokines, respectively. The contents of all the flasks sampled wassoluble in 5 ml of bidistilled water in less than 1 minute.

Example 7 Crossed Immunological Reactions of the Composition of F(ab′)₂Against Bothrops asper and Crotalus durissus durissus (AntiviperinComposition) with the Venom of Other Species and Genera

For each of the below mentioned snakes species, eight different amountsof the antiviperin composition together with a fixed amount of venom(equivalent to 5 LD₅₀) and saline solution to fix the volume, wereincubated for 1 hr at 37° C. After that, eight groups of 5 mice (Balb/c)of an average weight of 15 g were injected intra peritoneally with afixed volume of 700 μl, one group for each amount of the antiviperincomposition. Mortality of mice was read after 24 hours.

Experimental data of percentage of mortality vs. logarithm of volumeadded (of the antiviperin composition) were adjusted by a non-linealregression with GraphPad PRISM software (GraphPad Software Inc., SanDiego Calif.) and the Effective Doses 50% (ED50) (defined as theantivenom/venom proportion in which the lethal activity is reduced to50% compared to the effect of the same amount of venom alone) werecalculated for the antiviperin composition (antivenom) against each ofthe venoms. Table I shows the for the antiviperin composition againsteight snakes species from 4 different species/ three different genera.

TABLE 1 Effective Doses 50% (ED₅₀) expressed as milligrams (mg), for 5lethal doses 50% (LD₅₀) of the venom from different snake species. ED₅₀of the antiviperin Venom (5.0 LD₅₀) from: composition (mg) Bothropsalternatus 5.88 Bothrops neuwedii 1.62 Bothrops jararaca 2.09 Bothropsmoojeni 0.98 Agkistrodon bilineatus 4.39 Bothrops asper 0.61 Crotalusdurissus durissus 1.73 Bothrops nummifer 7.05

The above results show that the antiviperin composition containing atleast two F(ab′)₂ against Bothrops and Crotalus genera neutralizesspecies from genera Bothrops, Crotalus and Agkistrodon.

Example 8 Determination of Antiviperin Composition Titer Against 16Snakes Species by ELISA

ELISA plates were prepared with venom from 16 snake species, reactedwith the antiviperin composition (against venom from Bothrops andCrotalus genera), developed and read at 405 nm. Experimental data forAbsorbance at 405 nm vs. logarithm of dilution factor (of theantiviperin composition) were adjusted by a non-lineal regression withGraphPad PRISM software (Graphpad Software Inc., San Diego, Calif.) andthe Titer (defined as the dilution factor at which half of the maximumresponse is reached) for the antiviperin composition against each venomwere calculated and are represented in Table 2.

TABLE 2 Antiviperin composition titer against several snakes speciesfrom four different genera. Snake specie Titer Bothrops atrox asper 2177Bothrops asper 2842 Bothrops nummifer  890 Bothrops undulates 1802Crotalus basiliscus 3073 Crotalus durissus durissus 1226 Agkistrodonbilineatus bilineatus 1120 Crotalus scutulatus 2884 Bothrops alternates1774 Bothrops neuwiedii 3052 Bothrops moojeni 2230 Bothrops jararaca2572 Bothrops jararacussu 2916 Bothrops ammodytoides 1438 Crotalusdurissus terrificus 1040 Lachesis muta stenophry 2183

The results demonstrate that F(ab′)₂ contained in the antiviperincomposition of the present invention neutralizes venoms from snakes fromthe genera Bothrops, Crotalus, Lachesis and Agkistrodon.

The above examples have been depicted solely for the purpose ofexemplification and are not intended to restrict the scope orembodiments of the invention. The invention is further illustrated withreference to the claims which follow hereto.

1. A pharmaceutical composition comprising polyclonal F(ab′)₂ antibodyfragments substantially free from albumin and whole antibodies andsubstantially free of pyrogens, wherein said composition binds andneutralizes a purified antigenic molecule or mixture of antigenicmolecules found in the venom of a scorpion of the species Centruroideslimpidus, and wherein the F(ab′)₂ antibody fragments are obtained by themethod which comprises: (a) contacting a source of antibody with pepsinunder conditions to prepare an antibody digest containing F(ab′)₂fragments and being substantially free of unhydrolyzed antibodies; (b)treating said antibody digest by two steps of ammonium sulfateprecipitation, i) one step at about 16% to about 22% weight by volumeammonium sulfate; and ii) another step at about 32% to about 38% weightby volume of ammonium sulfate.
 2. The composition of claim 1, furthercomprising a pharmaceutically acceptable carrier.
 3. A pharmaceuticalcomposition comprising polyclonal F(ab′)₂ antibody fragmentssubstantially free of albumin, viral particles, whole antibodies andsubstantially free of pyrogens, wherein said composition binds andneutralizes a purified antigenic molecule or mixture of antigenicmolecules found in the venom of a scorpion, and wherein the F(ab′)₂antibody fragments are obtained by the method which comprises: (a)generating a source of antibodies from an animal that has been immunizedwith a complex mixture of antigenic molecules found in the venom of ascorpion of the species Centruroides limpidus; (b) contacting saidsource of antibodies with pepsin under conditions to prepare an antibodydigest containing F(ab′)₂ fragments wherein said digest is substantiallyfree of unhydrolyzed antibodies; (c) treating said antibody digest bytwo steps of ammonium sulfate precipitation, i) one step at about 16% toabout 22% weight by volume ammonium sulfate; and ii) another step atabout 32% to about 38% weight by volume of ammonium sulfate to therebyobtain a suspension containing F(ab′)₂ fragments substantially free ofwhole antibodies; (d) centrifuging said suspension to produce acomposition comprising a paste of F(ab′)₂ fragments and a supernatant;and (e) removing said supernatant from the composition produced in step(d).
 4. The composition of claim 3, wherein said composition furthercomprises a pharmaceutically acceptable carrier.
 5. A compositioncomprising polyclonal F(ab′)₂ antibody fragments substantially free fromalbumin and whole antibodies and substantially free of pyrogens, whereinsaid composition binds and neutralizes a purified antigenic molecule ormixture of antigenic molecules found in the venom of a scorpion of thespecies Centruroides limpidus, and wherein the F(ab′)₂ antibodyfragments are obtained by the method which comprises: (a) contacting asource of antibody with pepsin under conditions to prepare an antibodydigest containing F(ab′)₂ fragments and being substantially free ofunhydrolyzed antibodies; (b) treating said antibody digest by two stepsof ammonium sulfate precipitation, i) one step at about 16% to about 22%weight by volume ammonium sulfate; and ii) another step at about 32% toabout 38% weight by volume of ammonium sulfate.
 6. The composition ofclaim 1, wherein said scorpion is Centruroides limpidus tecomanus. 7.The composition of claim 1, wherein said scorpion is Centruroideslimpidus limpidus.
 8. The composition of claim 5, wherein said scorpionis Centruroides limpidus tecomanus.
 9. The composition of claim 5,wherein said scorpion is Centruroides limpidus limpidus.
 10. Apharmaceutical composition comprising polyclonal F(ab′)₂ antibodyfragments substantially free from albumin and whole antibodies andsubstantially free of pyrogens, wherein said composition binds andneutralizes a mixture of antigenic molecules found in the venom ofCentruroides noxius, Centruroides limpidus limpidus, Centruroideslimpidus tecomanus and Centruroides suffussus suffussus, and wherein theF(ab′)₂ antibody fragments are obtained by the method which comprises:(a) contacting a source of antibody with pepsin under conditions toprepare an antibody digest containing F(ab′)₂ fragments and beingsubstantially free of unhydrolyzed antibodies; (b) treating saidantibody digest by two steps of ammonium sulfate precipitation, i) onestep at about 16% to about 22% weight by volume ammonium sulfate; andii) another step at about 32% to about 38% weight by volume of ammoniumsulfate.
 11. A composition comprising polyclonal F(ab′)₂ antibodyfragments substantially free from albumin and whole antibodies andsubstantially free of pyrogens, wherein said composition binds andneutralizes a mixture of antigenic molecules found in the venom ofCentruroides noxius, Centruroides limpidus limpidus, Centruroideslimpidus tecomanus and Centruroides suffussus suffussus, and wherein theF(ab′)₂ antibody fragments are obtained by the method which comprises:(a) contacting a source of antibody with pepsin under conditions toprepare an antibody digest containing F(ab′)₂ fragments and beingsubstantially free of unhydrolyzed antibodies; (b) treating saidantibody digest by two steps of ammonium sulfate precipitation, i) onestep at about 16% to about 22% weight by volume ammonium sulfate; andii) another step at about 32% to about 38% weight by volume of ammoniumsulfate.
 12. A pharmaceutical composition comprising polyclonal F(ab′)₂antibody fragments substantially free of albumin, viral particles, wholeantibodies and substantially free of pyrogens, wherein said compositionbinds and neutralizes a purified antigenic molecule or mixture ofantigenic molecules found in the venom of a scorpion, and wherein theF(ab′)₂ antibody fragments are obtained by the method which comprises:(a) generating a source of antibodies from an animal that has beenimmunized with a complex mixture of antigenic molecules found in thevenom of Centruroides noxius, Centruroides limpidus limpidus,Centruroides limpidus tecomanus and Centruroides suffussus suffussus;(b) contacting said source of antibodies with pepsin under conditions toprepare an antibody digest containing F(ab′)₂ fragments wherein saiddigest is substantially free of unhydrolyzed antibodies; (c) treatingsaid antibody digest by two steps of ammonium sulfate precipitation, i)one step at about 16% to about 22% weight by volume ammonium sulfate;and ii) another step at about 32% to about 38% weight by volume ofammonium sulfate to thereby obtain a suspension containing F(ab′)₂fragments substantially free of whole antibodies; (d) centrifuging saidsuspension to produce a composition comprising a paste of F(ab′)₂fragments and a supernatant; and (e) removing said supernatant from thecomposition produced in step (d).